For scanning electron microscopy analysis, 25% glutaraldehyde was diluted by phosphate buffer (PH = 7.0) into 3% final fixation solution, and then leaf tissue samples were fixed in 3% glutaraldehyde overnight. The tissues were washed and dehydrated with gradient ethanol. Tissues samples were dried and coated with gold and examined under a Quanta 250 FEG scanning electron microscope at an accelerating voltage of 5 kV (FEI, Hillsboro, OS, USA). For phase contrast microscopy analysis, fully expanded adult leaves were detached from wild-type, first fixed in ethanol:acetic acid (6:1) solution for 2 h twice in total at room temperature. The tissue samples were treated by chloraldurate:glycerol:water (8:1:3) solution, and examined by the fluorescence microscope with optical filter (Nikon NI-SS, Tokyo, Japan).
Ni ss
Manufactured by Nikon
Sourced in Japan
The NI-SS is a scientific instrument designed for specialized laboratory applications. It serves as a core component in various research and analysis workflows. The device's primary function is to facilitate precise measurements and data collection within controlled environments. Technical details and specific use cases are not included in this description.
Automatically generated - may contain errors
Lab products found in correlation
Neutral balsam, by Sinopharm (2 mentions)
Live dead cell staining kit, by Abcam (2 mentions)
Quanta 250 feg scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Ab7817, by Abcam (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Leica tcs sp5 spectral laser scanning confocal microscope, by Leica camera (1 mentions)
7 protocols using ni ss
1
Leaf Tissue Microscopy Protocol
2
Visualizing Cell Adhesion on 3D Scaffolds
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To investigate cell behavior, we observed the focal adhesion of MC3T3-E1 cells cultured in α-MEM medium for 5 h on the scaffolds.
Staining actin filaments with phalloidin can detect the actin cytoskeleton of MC3T3-E1 cells on the 3D scaffolds. For actin cytoskeleton and focal adhesion identification, 1 × 105 cells/well were seeded on the scaffolds and incubated for 5 h to adhere to each surface of scaffold. The MC3T3-E1 cells on the scaffolds were washed in PBS and fixed in 4% PFA for 15 min. The fixed cells were permeabilized with 0.1% Triton X-100, blocked with 1% BSA for 30 min and incubated overnight at 4 °C with anti-vinculin antibody and anti-paxillin antibody diluted 1:50. After overnight, the cells on the scaffolds were washed thrice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:1000) for 2 h. Then, the scaffolds were washed in PBS and mounted in mounting medium with DAPI and phalloidin mixed at 1:1. Fluorescence of vinculin and paxillin (green), actin fibers (red), and nuclei (blue) were imaged with a fluorescence microscope (NI-SS, Nikon, Tokyo, Japan).
Staining actin filaments with phalloidin can detect the actin cytoskeleton of MC3T3-E1 cells on the 3D scaffolds. For actin cytoskeleton and focal adhesion identification, 1 × 105 cells/well were seeded on the scaffolds and incubated for 5 h to adhere to each surface of scaffold. The MC3T3-E1 cells on the scaffolds were washed in PBS and fixed in 4% PFA for 15 min. The fixed cells were permeabilized with 0.1% Triton X-100, blocked with 1% BSA for 30 min and incubated overnight at 4 °C with anti-vinculin antibody and anti-paxillin antibody diluted 1:50. After overnight, the cells on the scaffolds were washed thrice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:1000) for 2 h. Then, the scaffolds were washed in PBS and mounted in mounting medium with DAPI and phalloidin mixed at 1:1. Fluorescence of vinculin and paxillin (green), actin fibers (red), and nuclei (blue) were imaged with a fluorescence microscope (NI-SS, Nikon, Tokyo, Japan).
3
Vitelline Cell Phenolic Substance Staining
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The phenolic substances in mature vitelline cells can be positively stained with Fast Red B [34 (link)]. In the present study, Fast Red B was substituted by Fast Blue B (Sinopharm Chemical Reagent Co., Ltd, China), a related diazonium salt which exhibits a similar staining profile [35 (link)]. Female worms were fixed in 70% ethanol for at least 24 h, and then stained with filtered 1% Fast Blue B solution for 1 min. The worms were dehydrated through an ethanol gradient and mounted in neutral balsam (Sinopharm Chemical Reagent Co., Ltd, China). The staining of vitelline cells was evaluated by light microscopy using a compound binocular microscope (Nikon NI-SS, Japan) under 40×.
4
Immunofluorescence Staining of α-SMA
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Cells were washed three times in 1× PBS, fixed in 3% formaldehyde (DUKSAN, Gyeonggi-do, Republic of Korea) in cold PBS at 4°C for 30 min, rinsed three times in 1× PBS, and permeabilized with 0.5% Triton X-100 in 1× PBS at room temperature for 10 min. Cells were further rinsed three times in 1× PBS for 5 min each, blocked with 5% BSA in PBS at room temperature for 1 h, and stained with α-SMA antibody (1:200; ab7817; Abcam) for 3 h at room temperature followed by five rinses in 1× PBS for 5 min each. The cells were incubated with goat anti-mouse IgG conjugated to Alexa 488 (1:1,000; A11001; Invitrogen) for 1 h at room temperature in the dark and rinsed in 1× PBS five times for 5 min each. Coverslips were mounted using SlowFade Gold antifade reagent with DAPI (S36938; Invitrogen). Fluorescence images were visualized under an optical microscope (Ni-SS; Nikon Instruments, Melville, NY, USA). Positive immunofluorescence was quantified on the basis of optical intensity using NIS Elements BR software. Representative images of α-SMA (Alexa 488; excited at 488 nm; green) and DAPI (nuclei; excited at 405 nm; blue) staining were taken, and the average intensity in more than five fields from at least three independent experiments was graphed.
5
Staining Protocols for Parasitic Worm Analysis
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For Fast Blue B staining, female worms were fixed in 70% ethanol for ≥24 h. After staining with filtered 1% (w/v) Fast Blue B solution, they were dehydrated through an ethanol gradient from 70 to 100%, and then mounted in neutral balsam (Sinopharm Chemical Reagent Co., Ltd, China)66 (link). Staining of vitelline cells was evaluated using light microscopy (Nikon NI-SS, Japan). For hydrochloric carmine staining, worms were separated by sex and fixed in AFA (alcohol 95%, formalin 3%, glacial acetic acid 2%). Worms were stained with hydrochloric carmine for 30 min and destained in acidic 70% ethanol. After sequential dehydration in graded ethanol (70, 90, 100%), worms were mounted on glass slides with neutral balsam. Confocal images were taken with a Leica TCS-SP5 Spectral Laser Scanning Confocal Microscope (Leica, Germany) using a 488-nm He/Ne laser.
6
Cell Viability Evaluation of MC3T3-E1 Cells on Scaffolds
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The cell viability was evaluated by staining cells using a live/dead cell staining kit (Biovision, Milpitas, CA, USA). The MC3T3-E1 cells were seeded on the scaffolds at a density of 5 × 105 cells/well on scaffolds in 12-well plates and were cultured on scaffolds for two days in CO2 incubator.
After two days, the culture medium was removed from the scaffolds and the scaffolds were washed twice with PBS. Then, 1 mL of the staining solution (1 mM cell-permeable green fluorescent dye and 2.5 mg/mL of propidium iodide) per well and the culture plates were returned to the incubator for 20 min. Live (green) and dead cells (red) were imaged with a fluorescence microscope (NI-SS, Nikon, Tokyo, Japan).
After two days, the culture medium was removed from the scaffolds and the scaffolds were washed twice with PBS. Then, 1 mL of the staining solution (1 mM cell-permeable green fluorescent dye and 2.5 mg/mL of propidium iodide) per well and the culture plates were returned to the incubator for 20 min. Live (green) and dead cells (red) were imaged with a fluorescence microscope (NI-SS, Nikon, Tokyo, Japan).
7
Cell Viability Assay on MC3T3-E1 Cells
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Cell viability was assessed using a Molecular Probes Live-Dead cell staining kit (Biovision, Milpitas, CA, USA). MC3T3-E1 cells were seeded at a density of 3×10 5 cells/mL on scaffolds in 48-well plates. After 2 days incubation, the culture media was removed from the wells and cells/scaffolds were rinsed three times with DPBS. Then staining solution (1 mM live-dye and 2.5 mg/mL of propidium iodide), 0.25 mL per well, was added, and the plates were cultured in an incubator for 20 min. Live cells (green) and dead cells (red) were visualized by fluorescence microscopy (NI-SS, Nikon, Tokyo, Japan) using a band-pass filter.
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